Abstract Postnatal expression of chondroitin sulfateproteoglycans was studied in the rat thalamus by immu-nocytochemistry and Western immunoblotting tech-niques with monoclonal antibodies that recognize carbo-hydrate epitopes (clones CS-56, 1-B-5, 2-B-6). The com-plex of the results shows that these antibodies recognizemostly nonoverlapping molecules whose expression isregulated during postnatal development. Chondroitin sul-fate proteoglycans, recognized by antibody CS-56, andhyaluronan, identified by antibody 1-B-5 after hyaluroni-dase digestion, are abundant in the neuropil of most tha-lamic nuclei at the perinatal stage and progressively de-crease during the second week of life, attaining levelsbarely detectable by immunocytochemistry at the end ofthe third week. In adult thalamus, chondroitin sulfateproteoglycans of high molecular mass, bearing glycos-aminoglycans unsulfated in the linking region, and rec-ognized by antibody 1-B-5 are confined to perineuronalnets around neurons chiefly localized in thalamic reticu-lar nucleus. The immunoreactvity for antibody 2-B-6,specific for chondroitin-4-sulfate, is low at the perinatalstage and is not detectable in adult thalamus. Double-im-munolabeling has shown that, along the rostrocaudal ex-tension of reticular nucleus, the most developed perineu-ronal nets are associated with a subset of neurons ex-pressing calretinin, and not with parvalbumin-positiveneurons, which represent the largest neuronal populationof the nucleus. The distribution of perineuronal nets sup-ports the presence, in thalamic reticular nucleus, of neu-ronal subpopulations with different morphological andphysiological features.

Keywords Calretinin · Electron microscopy · Glycosaminoglycans · Parvalbumin · Thalamic reticularnucleus · Rat (Wistar)

Introduction

Chondroitin sulfate proteoglycans (CSPGs) are the mostabundant components of the extracellular matrix of themammalian brain, encompassing at least 16 differenttypes of molecules expressed at different developmentalstages (Herndon and Lander 1990). Extensive experi-mental evidence indicates that these molecules, many ofwhich belong to the lectican family, are involved in sev-eral developmental processes, regulating cell prolifera-tion and differentiation and providing either inhibitory orpermissive cues for axon outgrowth (for reviews, seeLetournau et al. 1994; Margolis and Margolis 1994;Oohira et al. 1994; Yamaguchi 2000; Hartmann andMaurer 2001). Previous studies (Aquino et al. 1984; Flaccus et al. 1991; Rauch et al. 1991; Oohira et al.1994; Maeda et al. 1995; Miller et al. 1995; Fernaud-Espinosa et al. 1996; Köppe et al. 1997; Hoffman-Kim et al. 1998) have shown that, during brain development,a diffuse and relatively abundant extracellular matrixcontains complex combinations of CSPGs, whose chem-ical microheterogeneity is responsible for different bio-logical effects.

Immunohistochemistry and lectin cytochemistry haverevealed that, in the gray matter of adult brain, CSPGsare mainly concentrated in lattice-like envelopes, termedperineuronal nets, around cell bodies and proximal den-drites of distinct neuronal populations (for reviews, seeCelio and Blümcke 1994; Celio et al. 1998; Matsui et al.1999; Yamaguchi 2000). The presence of perineuronalnets does not identify classes of neurons homogeneousfor the expression of other neurochemical markers butfrequently corresponds to functional cortical areas andsubcortical regions (Hockfield et al. 1983; Hendry et al.1988; Sur et al. 1988; Crabtree and Kind 1993; Preuss et al. 1998; Gray et al. 1999; Pimenta et al. 2001).

The distribution of CSPGs in the rat thalamus has not been studied in detail, although previous studies have shown that, in adult brain, perineuronal nets aremainly concentrated in the thalamic reticular nucleus(Rt; Bertolotto et al. 1990, 1991, 1996; Seeger et al.

L. Vitellaro-Zuccarello (✉ ) · A. Meroni · A. Amadeo · S. De BiasiSezione di Istologia e Anatomia Umana, Dipartimento di Fisiologia e Biochimica generali, Via Celoria 26,20133 Milan, Italye-mail: laura.vitellaro@unimi.itTel.: +39-2-70644303, Fax: +39-2-2362808

Cell Tissue Res (2001) 306:15–26DOI 10.1007/s004410100425

R E G U L A R A R T I C L E

L. Vitellaro-Zuccarello · A. Meroni · A. AmadeoS. De Biasi

Chondroitin sulfate proteoglycans in the rat thalamus: expression during postnatal development and correlation with calcium-binding proteins in adultsReceived: 29 January 2001 / Accepted: 11 May 2001 / Published online: 3 July 2001© Springer-Verlag 2001

1994; Brückner et al. 1996). In the present investigation,we have used immunocytochemical and immunoblottingtechniques to study the expression of CSPGs in the thal-amus of the rat during postnatal development. Moreover,with double-labeling techniques, we have investigatedthe distribution of perineuronal nets in adult thalamus inrelation to the expression of the calcium-binding proteinsthat identify functionally distinct populations of neurons(Celio 1990; Arai et al. 1994; Winsky et al. 1992; Lizieret al. 1997).

Materials and methods

Animals

Postnatal rats (Wistar; Charles River, Calco, Italy), ranging fromthe day of birth (defined as postnatal day 0, P0) to the end of thethird postnatal week, and adult rats weighing about 250 g wereused. The University of Milan approved the care and handling ofanimals in accordance with all NIH guidelines.

Antibodies

The following mouse monoclonal antibodies (MAbs) were used:MAb CS-56 (IgM, Sigma, St. Louis, Mo.), which recognizes the6-sulfated and, with lower affinity, the 4-sulfated forms of an asyet poorly characterized epitope present in the interior of nativechondroitin sulfate (Avnur and Geiger 1984; Sorrel et al. 1993);MAbs 1-B-5 and 2-B-6 (IgG, ICN Pharmaceuticals, Costa Mesa,Calif.), which recognize oligosaccharide stubs resulting from thedigestion of CSPGs with chondroitinase ABC and containing aterminal unsaturated hexuronic acid adjacent either to unsulfatedN-acetylgalactosamine (1-B-5) or to 4-sulfated N-acetylgalactos-amine (2-B-6; Caterson et al. 1985). For double-labeling experi-ments, MAb 1-B-5 was used in combination with either polyclonalanti-calretinin or polyclonal anti-parvalbumin antibodies (SWant,Bellinzona, Switzerland).

Electrophoresis and Western blot analysis

Adult and 4-day-old rats were anesthetized by intraperitoneal in-jection of chloral hydrate (4%, 1 ml/100 g body weight) and trans-cardially perfused with ice-cold 50 mM TRIS-HCl buffer, pH 6.5,containing 0.15 M NaCl, 2 mM EDTA, 100 µM N-ethylmaleimi-de, 500 µM phenylmethylsulfonyl fluoride. A thick brain slice in-cluding the thalamus was homogenized in a glass-Teflon homoge-nizer in five volumes of ice-cold perfusion buffer to which1 µg/ml pepstatin and leupeptin had been added. After removal ofthe low speed pellet (800 g for 10 min), the supernatant was cen-trifuged at 100,000 g for 30 min, and the resulting supernatant wascollected.

A few aliquots of the homogenate were dialyzed against 0.1 MTRIS-HCl, pH 7.2, containing 0.1 M Na acetate and digested withchondroitinase ABC (Sigma, 5 mU/100 µg protein) in the presenceof proteinase inhibitors for 2 h at 37°C.

After separation by SDS-polyacrylamide gel electrophoresis(SDS-PAGE; 5% separating gels), proteins were transferred tonitrocellulose. The blots, with or without previous digestion withchondroitinase ABC, were sequentially probed with primary antibodies and the appropriate horseradish-peroxidase-labeledsecondary antibody. Immunoreactivity was detected by using theBM chemiluminescence Western blotting kit (Boehringer, Mann-heim, Germany) according to the manufacturer’s instructions.Chemiluminescent bands were recorded on Kodak X-Omat ARfilms.

Light and confocal microscopy

At least three animals were used at each selected age (P0, P2, P5,P7, P9, P11, P15, P18, P20, adult). The animals, under deep anes-thesia, were perfused transcardially with 1% paraformaldehyde in0.1 M phosphate buffer (PB), pH 7.2, followed by a mixture of 4%paraformaldehyde and 0.5% glutaraldehyde in the same buffer.The brains were then dissected out, postfixed in 4% paraformalde-hyde for 12 h, and embedded in paraffin or serially cut in coronalsections (50 µm) on a Vibratome throughout the rostrocaudal ex-tent of the thalamus. Selected paraffin or vibratome sections wereincubated in primary antibody. Antigen-antibody reaction was re-vealed by the standard ABC-peroxidase method (Hsu et al. 1981)with the Vectastain ABC kit (Vector Laboratories, Burlingame,Calif.) and diaminobenzidine tetrahydrochloride (DAB) as a chro-mogen. Prior to immunoreaction with 1-B-5 and 2-B-6 MAbs,sections were subjected to enzymatic digestion with 0.2 U/mlchondroitinase ABC in 0.1 M TRIS-HCl, pH 7.2, containing0.1 M Na acetate and 20 µg/ml bovine serum albumin (BSA;75 min at 37°C). In other experiments, sections were digested with100 U/ml Streptomyces hyaluronidase (Sigma) in 0.05 M acetatebuffer, pH 5 (3 h at 37°C), prior to incubation with MAb 1-B-5.The incubation media for enzyme digestion always contained acocktail of protease inhibitors.

Control sections were submitted to the same immunocyto-chemical staining sequence, except that the primary antibody wasomitted or replaced with equivalent concentrations of unrelatedIgM or IgG of the same subclass.

For double-labeling experiments, sections were incubated witha mixture of MAb 1-B-5 and anti-parvalbumin or anti-calretininserum. Sections were subsequently incubated with a mixture of

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Fig. 1 Immunoblot analysis of homogenates from P4 (P4) andadult (Ad) brain not subjected (A) or subjected (B) to digestionwith chondroitinase ABC before SDS-PAGE. The amount of pro-tein loaded per lane was 100 µg (arrows gel top). Molecularweights are in kDa

the appropriate fluorescein-isothiocyanate-conjugated or liss-amine-rhodamine-conjugated secondary antibodies (Jackson Im-munoresearch, West Grove, Pa.). Double-labeled vibratome sec-tions were examined under a TCS NT confocal laser scan micro-scope (Leica Lasertechnik, Heidelberg, Germany).

Immunoelectron microscopy

Brain coronal sections, fixed as for light microscopy immunocyto-chemistry, were permeabilized by a mild ethanol treatment (10%,25%, and 10%; 5 min each) or with 0.2% saponin in PB contain-ing 1% BSA (3 changes; 10 min each). Sections were subsequent-ly immunolabeled with MAb CS-56 or 1-B-5 according to theABC method, postfixed with 1% osmium tetroxide, and flat-em-

bedded in Epon-Spurr. Ultrathin sections of the regions of interestwere cut on a Reichert ultramicrotome and examined (unstained orcounterstained with lead citrate only) with a Zeiss 902 electronmicroscope. Electron-spectroscopic imaging procedures were rou-tinely employed to improve contrast.

Results

Western blot analysis

Both 1-B-5 and 2-B-6 (Fig. 1A) MAbs recognized highmolecular weight components (over 400 kDa) only inadult brain homogenates. An additional band of approxi-

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Fig. 2A, B Light micrographsof coronal vibratome sectionsof rat thalamus showing thedistribution of CS-56 immuno-reactivity during the first weekof life. ABC-DAB method.A Intermediate part of the thal-amus at P1. B Detail of the dis-tribution of CS-56 immunore-activity in Rt and ventroposte-rior nucleus at P5. Asterisksand arrows indicate, respec-tively, neuronal cell bodies andtransversely sectioned nerve fibers outlined by immunoreac-tivity (CL central lateral nucle-us, LG lateral geniculate nucle-us, Po thalamic posterior com-plex, Rt reticular nucleus,VP ventroposterior nucleus).Bars A 500 µm, B 50 µm

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Fig. 3 Light micrographs of coronal paraffin sections of the thala-mus immunostained with MAb 1-B-5. A–C ABC-DAB method.D–F Immunofluorescence. The micrographs show the distributionof immunoreactivity in Rt at P15 (A) and in adult thalamus (B–F).Thicker perineuronal nets (arrows) are recognizable along the me-dial and lateral aspects (B) and in the ventromedial corner (C) of

the intermediate part of Rt. D Detail of perineuronal nets in Rt.E posterior sector of Rt. F Distribution of the immunoreactivity inthe medial part of ventroposterior nucleus. ic Internal capsule,Rt reticular nucleus, VPL lateral part of ventroposterior nucleus.Bars A–C 200 µm, D 10 µm, F 50 µm

mately 95 kDa was recognized by MAb 1-B-5 in both P4and adult brain.

Digestion of brain extracts with chondroitinase ABCprior to electrophoresis increased the mobility of thecore proteins recognized by 1-B-5 and 2-B-6 antibodies(Fig. 1B). The intensities of all bands were higher in theadult than in P4 samples. Both antibodies identified atleast two bands with molecular weight over 400 kDa andwith similar electrophoretic mobility, whereas MAb 2-B-6 recognized additional bands of approximately 400,150, and 120 kDa.

The CS-56 antibody revealed several components ofmolecular weights between 95 and 150 kDa, and only inadult brain fraction, an additional component of about400 kDa (Fig. 1A). Two major bands of approximately100 kDa appeared to be more intense in P4 brain than inadult brain fractions. No immunoreactive bands wererecognized by this antibody in blots submitted to prelim-inary digestion with chondroitinase ABC.

Immunocytochemistry

The pattern of labeling obtained with all antibodies washighly reproducible in animals of comparable age. No im-munostaining was found in control sections in which theprimary antibody was replaced with unrelated IgM or IgG.

In the first postnatal week, MAbs CS-56 and 1-B-5gave a similar labeling pattern consisting of finely granularmaterial forming a delicate meshwork in the neuropil of allthalamic nuclei (Fig. 2A), whereas cell bodies were alwaysunstained (Fig. 2B). The intensity of labeling varied amongthe different thalamic nuclei whose boundaries were fre-quently outlined by the accumulation of immunoreactivematerial (Fig. 2A). The level of immunostaining was maxi-mum in the laterodorsal, lateroposterior, and lateral genicu-late nuclei and in most midline nuclei, and minimum in theventral posterolateral and ventroposterior nucleus (Fig. 2A)and in the anteroventral and anteromedial nuclei.

During the second postnatal week, immunoreactivitydecreased progressively. On P12, CS-56 immunostainingwas detectable only in the neuropil of Rt and of a fewmidline nuclei, and, after P15, it was no longer recogniz-able. Labeling for MAb 1-B-5 declined in most thalamicnuclei but remained intense in the neuropil of Rt and ofmost midline nuclei.

During the third postnatal week, except for a light anddiffuse positivity in the neuropil of midline nuclei, im-munoreactivity for MAb 1-B-5 was mainly concentratedin Rt, in which perineuronal staining became recogniz-able around a few neurons at about P15 (Fig. 3A). At theend of the third week, the labeling pattern of Rt attainedthe adult configuration (Figs. 3B–E), and intensely la-beled perineuronal nets surrounded the perikarya andproximal dendrites of several neurons.

In adult thalamus, the distribution of perineuronalnets and their labeling intensity varied along the rostro-caudal axis of Rt (Fig. 4). The rostral and caudal(Fig. 3E) parts of Rt and the ventromedial corner in the

intermediate sector of the nucleus (Fig. 3C) displayedstrong staining of perineuronal nets and of the interven-ing neuropil. Moreover, the thickest and most intenselylabeled perineuronal nets were concentrated along themedial and, to a lesser extent, lateral edges of the nucle-us (Figs. 3B, E, 5A, C, E). Conversely, perineuronal netswere very thin or absent in the central part of the inter-mediate sector of Rt (Fig. 3B).

Outside Rt, a small cluster of thin perineuronal netswas consistently observed in the dorsal part of the centrallateral nucleus. Sparse and less intensely labeled perineu-ronal nets were also detected in the ventroposterior(Fig. 3F) and posterior nuclei, whereas diffuse faint neuro-pilar immunoreactivity persisted in most midline nuclei.

The distribution of chondroitin 4-sulfate, identified byMAb 2-B-6, was notably different from that of the otherantigens examined, since, only at the perinatal stage, thinmeshworks of immunoreactive material were recognizableonly in the neuropil of the lateroposterior nucleus and ofthe midline nuclei. With increasing age, even this positiv-ity decreased to a very pale and diffuse background. Withthis antibody, perineuronal nets were never observed inthe thalamus, although, in the same sections, they were in-tensely labeled throughout the cerebral cortex.

Pretreatment of sections with chondroitinase ABC be-fore immunostaining with MAb CS-56 abolished immu-noreactivity. Omission of chondroitinase ABC pretreat-ment abolished immunostaining with MAbs 1-B-5 and2-B-6. Pretreatment of sections with Streptomyces hyalu-ronidase before immunostaining with MAb 1-B-5 pro-duced an intense immunostaining of the neuropil of allthalamic nuclei only at the perinatal stage.

Double immunolabeling for perineuronal nets and calcium-binding proteins in adult thalamus

Since perineuronal nets are mainly concentrated in Rt,the study of their association with calcium-binding pro-

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Fig. 4 Schematic representation of the distribution of perineuro-nal nets at different rostrocaudal levels (from left to right) of adultRt (black and open dots indicate, respectively, the distribution ofthick and thin perineuronal nets)

teins has been focused on this nucleus. Previous studies(Celio 1990; Arai et al. 1994; Winsky et al. 1992; Lizieret al. 1997) have shown that most neurons of Rt containparvalbumin, whereas calretinin and calbindin have amore restricted distribution.

The results of our double-labeling experimentsshowed that the association between neuronal positivityfor parvalbumin and perineuronal nets was extended, butnot total. The most notable exceptions were representedby (1) the weak pericellular staining in the central part of the intermediate Rt (Fig. 3B) in which all neurons express parvalbumin (not shown); (2) the strong stainingof both perineuronal nets and the intervening neuropil in the ventral border of the rostral medial corner, an

area almost devoid of parvalbulmin-positive neurons(Fig. 5A, B).

In general, the most intense labeling of perineuronalnets and neuropil matched the distribution of calretinin-positive neurons, which were especially concentrated inthe rostral (Fig. 5C, D) and caudal sectors of Rt, in thedorsal cap of the intermediate part of the nucleus(Fig. 5E, F), and along the medial and lateral edges.However, even within these areas, not all calretinin-posi-tive neurons were surrounded by pericellular staining.

At high magnification, the distribution of immunoreac-tivity for MAb 1-B-5 around cell bodies and proximal den-drites of neurons positive for either parvalbumin or calreti-nin appeared to be similar (Fig. 6A–D). Moreover, in many

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Fig. 5 Analysis by confocalmicroscope of vibratome sec-tions of adult Rt double-labeledwith MAb 1-B-5 (A, C, E) andwith either anti-parvalbumin(B) or anti-calretinin (D, F) antibodies. Insets Position atwhich coronal sections weretaken along the rostrocaudalaxis of the nucleus.A–D Ventromedial corner ofthe anterior part of Rt.E, F Rostral pole of the inter-mediate sector of Rt. AM An-teromedial nucleus, Po thalam-ic posterior complex, Rt reticu-lar nucleus. Bars 100 µm (C same magnification as A)

neuropil regions, 1-B-5 immunoreactivity associated withdendrite surfaces produced dense reticulate staining(Fig. 6A, C). Outside Rt, a correlation between perineuro-nal nets and calretinin was found in neurons of the dorsalpart of central lateral nucleus and in the midline nuclei.

Immunoelectron microscopy

At early postnatal stages, the immunoreactivity forMAbs CS-56 and 1-B-5 was concentrated around the

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Fig. 6 Confocal micrographs showing, at higher magnification,perineuronal nets in adult Rt double-labeled with MAb 1-B-5 (A, C) and with either anti-parvalbumin (B) or anti-calretinin (D)antibodies. A, B Neurons located in proximity of the medial edgeof the intermediate sector of Rt (asterisk parvalbumin-positiveneuron lacking perineuronal net). C, D Dorsal cap of the interme-diate sector of Rt. Both calretinin-positive and negative (asterisks)neurons are enwrapped by perineuronal nets (N neuropil). Bars10 µm

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Fig. 7 A Electron micrograph of Rt neuropil at P5 showing nerveprocesses outlined by immunoreactivity for MAb CS-56. B, CElectron micrographs of neuronal cell bodies (CB) in adult Rt la-beled with MAb 1-B-5 after mild ethanol permeabilization (ar-rowheads immunoreactivity outlining a cell body). In C, labeledastrocytic processes (asterisks) are adjacent to an unlabeled terminal making an asymmetric axosomatic synapse (triangle).

D, E Electron micrographs of neuronal cell bodies (CB) in adultRt (D) and in central lateral nucleus (E) labeled with MAb 1-B-5after saponin permeabilization. Immunoreactivity is presentaround cell bodies (arrowheads), in Golgi complexes (G), and insmall vesicles (arrow). C Capillary, RER rough endoplasmic reticulum. Counterstaining with lead citrate. Bars A, C 0.5 µm,B, D 1 µm, E 3 µm

plasma membranes of nerve fibers, terminals (Fig. 7A),and perikarya. In adult Rt, perineuronal labeling forMAb 1-B-5 outlined perikaryal and dendritic plasmamembranes, being interrupted by unlabeled synapticcontacts (Figs. 7B, C, 8). MAb 1-B-5 labeling was foundinside and around astrocytic processes close to neuronalcell bodies (Fig. 7C). Perivascular astrocytes were al-ways unlabeled (Fig. 7E). When tissues were permeabili-zed with saponin, immunoreactivity was also detected inthe Golgi apparatus and in small vesicles clustered nearthe inner aspect of neuronal plasma membranes(Fig. 7D), but not in astrocytic cell bodies. MAb 1-B-5immunoreactivity, although less intense and sparser,showed a similar pattern of distribution in the dorsal partof central lateral nucleus (Fig. 6E) and in the ventropos-terior nucleus.

Discussion

The distribution of CSPGs in the central nervous systemhas so far been investigated by immunocytochemistrywith two types of antibodies. The first type recognizesthe protein core and provides a selective identification ofone molecular species. Conversely, antibodies of the sec-

ond type, directed against the carbohydrate moieties,should have a broader specificity for several CSPGsbearing similar GAGs. Indeed, antibodies of the secondtype have been frequently used as general markers ofCSPGs (Miller et al. 1995; Hoffman-Kim et al. 1998).To address the question of the specificity of the antibod-ies (belonging to the second type) used in this investiga-tion, we carried out Western blotting experiments onhomogenates of whole brain at two representative ages(P4 and adult). The results show that MAbs CS-56, 1-B-5,and 2-B-6 identify mostly nonoverlapping molecularspecies of CSPGs. These findings are in line with previ-ous observations (Sorrel et al. 1996) showing that GAGchains are not random polymers of disaccharides butconsist of domains that, in different CSPGs, are assem-bled in specific combinations. MAb CS-56 recognizesseveral components, most of which are expressed both atP4 and in adult brain and have apparent molecularweights between 95 kDa and 150 kDa. MAbs 1-B-5 and2-B-6 recognize higher-molecular-weight componentsthat are predominantly expressed in adult brain and thatseem to overlap partially, thus indicating that the sameCSPGs may exist in different sulfation forms. The ab-sence of immunoreactivity for MAb CS-56 in the high-molecular-weight components recognized by 1-B-5 and2-B-6 antibodies indicates that the GAG moieties ofthese molecules lack, or have undetectable levels, of the4/6-sulfated epitopes recognized by the former antibody.These findings agree with previous observations (Sorrelet al. 1996) indicating that MAb CS-56 cannot be con-sidered a generic marker for CSPGs.

The immunocytochemical analysis of the thalamushas shown that the distribution of CS-56 and 1-B-5 epi-topes during postnatal development is similar to that pre-viously described for the cerebral cortex (Miller et al.1995). With both antibodies, immunoreactivity is intenseafter birth and decreases progressively until it disappears(MAb CS-56) or becomes restricted to the perineuronalnets of distinct neuronal populations (MAb 1-B-5). Thepresence of components immunoreactive for MAb CS-56 observed in adult brain with Western blotting mightbe accounted for by the persistence of immunoreactivityin brain regions not considered in this study. Moreover,the masking of CS-56 epitopes may occur during tissuematuration and contribute to the decline of the immuno-reactivity observed in histologic sections.

With respect to MAb 1-B-5, the intense positivity dis-played only by the neonatal thalamus after digestionwith bacterial hyaluronidase suggests that hyaluronancontributes to the immunoreactivity observed at thisstage. This interpretation agrees with (1) the results ofWestern blotting experiments showing that CSPG(s) recognized by MAb 1-B-5 in neonatal brain are scanty,and (2) previous observations indicating that hyaluronanis abundant in brain at early developmental stages (Bignami et al. 1993). Moreover, it is known that MAb 1-B-5 also recognizes d-unsaturated disaccharidesof hyaluronan (Caterson et al. 1985) generated either bybacterial hyaluronidase, an enzyme that hydrolyses only

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Fig. 8 Electron micrograph of adult Rt neuropil showing a den-drite (D) outlined by immunoreactivity for MAb 1-B-5 and con-tacted by two terminals (asterisks) making symmetric axodendriticsynapses. Counterstaining with lead citrate. Bar 0.5 µm

hyaluronan (Derby and Pintar 1978), or by enzymes witha broader specificity, such as chondroitinase ABC.

On the basis of the results obtained with MAb 2-B-6,it appears that CSPGs with 4-sulfated initial GAGs con-tribute only in a limited fashion to the extracellular ma-trix of the immature thalamus and, at variance with thecerebral cortex, are no longer detectable in the adult or-gan. The latter finding indicates that regional differencesexist in the regulation of the enzymes of the GAG bio-synthetic pathway, probably in relation to specific func-tional requirements.

Overall, our observations confirm that CSPGs and hy-aluronan have a widespread distribution in the brain dur-ing the first postnatal week. In particular, these mole-cules could fill the extracellular spaces, which are widerin neonatal than in adult brain (Lehmenkühler et al.1993), thereby regulating the osmotic and swelling pres-sure of the tissue. Moreover, the highly hydrated envi-ronment provided by GAGs and their capacity to bindmorphogenetic factors (Celio and Blümcke 1994) mayfavor the processes of reorganization, remodeling, andmaturation still active in the thalamus during the firsttwo postnatal weeks (Frassoni et al. 1995; De Biasi et al.1996). The postnatal appearance of perineuronal nets atthe onset of the third week of life has been previouslyobserved with lectin histochemistry (Köppe et al. 1997)and corresponds to the time of acquisition of the matureneuronal phenotype (Spreafico et al. 1994; De Biasi et al. 1996, 1997).

Immunocytochemical and Western blotting results in-dicate that the CSPG(s) recognized by MAb 1-B-5 inadult thalamus, viz., the high-molecular-weight formsthat exhibit the prevalent expression in mature brain andwhose synthesis probably involves neurons, share fea-tures with CSPGs that contain Cat antigens (Hockfieldand McKay 1983; Hockfield et al. 1990; Fryer et al.1992; Lander et al. 1998) and that are related to aggrecan(Fryer et al. 1992).

Our immunocytochemical data extend previous obser-vations (Bertolotto et al. 1991, 1996) on the presence of1-B-5-positive perineuronal nets in adult rat thalamus. Inparticular, we have observed that differences exist in thelabeling intensity of perineuronal nets throughout therostrocaudal extension of Rt. This nucleus belongs to theventral thalamus and consists of a population of project-ing gamma-aminobutyric-acid-ergic cells (De Biasi et al.1986) that provide an inhibitory innervation to all nucleiof the dorsal thalamus (Steriade et al. 1997). The vastmajority of Rt neurons express parvalbumin and, to a lesser extent, calretinin and calbindin (Celio 1990;Winsky et al. 1992; Arai et al. 1994; Lizier et al. 1997).The present data demonstrate that the strongest labelingof the perineuronal nets and of the intervening neuropilmatches the distribution of calretinin-positive neurons.This association, however, does not correspond to func-tionally homogeneous sectors of Rt. Indeed, the most in-tense labeling of perineuronal nets has been detected inthe rostral pole of Rt, which is connected to limbic func-tions, and, more caudally, in the visual and acustic sec-

tors. Moreover, a strong pericellular staining is associat-ed with neurons distributed along the medial edge of thewhole nucleus. These marginal neurons, although be-longing to functionally different sectors, share a numberof features, such as their morphology (Spreafico et al.1991), their preferential expression of calretinin (Lizieret al. 1997; present study), and their distinctive expres-sion of neurofilament subunits and adhesive molecules(A. Amadeo, personal communication), and may repres-ent a subpopulation with distinct, although as yet un-known, functional properties. Interestingly, in the rat,neurons of the inner tier of the somatosensitive, visual,and acustic sectors of the Rt nucleus are connected withhigher order thalamic nuclei and with secondary corticalareas (Guillery et al. 1998). The correlation of perineuro-nal nets with calbindin has not been studied in the pres-ent investigation. However, previous studies have shown(Arai et al. 1994) that, in Rt, calbindin-positive neuronsare few and are concentrated in the central part of theventromedial corner, a region nearly devoid of pericellu-lar nets.

The cellular origin of CSPGs varies according to molecular species, since they can be of either neuronal orglial origin or both (Yamaguchi 2000). Our ultrastructuralobservations, showing the presence of 1-B-5 immunore-activity in astrocytic processes and in the Golgi complexof neurons, should be interpreted cautiously and are notsufficient to support an involvement of both cell types inthe biosynthesis of 1-B-5-immunoreactive molecules.

With regard to the function of perineuronal nets, at-tempts to correlate their presence with specific physio-logical properties of neurons have not to date given un-equivocal results (for reviews, see Celio and Blümcke1994; Celio et al. 1998), even if a growing body of evi-dence seems to indicate that they may be involved in theregulation of the ionic composition in the pericellular en-vironment (Spicer et al. 1996; Härtig et al. 1999). It hasbeen also proposed that perineuronal nets, consisting oftertiary complex of CSPGs, hyaluronan and tenascin-R,may play a role in the regulation of synaptic plasticityand stabilization (for reviews, see Celio et al. 1998; Yamaguchi 2000).

In conclusion, the results of this investigation confirmrecent findings on the architectural and neurochemicalcomplexity of Rt and the existence of complicate rela-tionships between perineuronal nets and neuronal physi-ology. Therefore, further work is needed to correlate theneurochemical and synaptic features of neurons en-wrapped by perineuronal nets with the composition ofthe pericellular matrix, whose microchemical complexityand heterogeneity probably imply diversified functionsin different regions of the central nervous system.

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